The Enterovirus Theory of Disease Etiology in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Critical Review
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REVIEW
published: 18 June 2021
doi: 10.3389/fmed.2021.688486
The Enterovirus Theory of Disease
Etiology in Myalgic
Encephalomyelitis/Chronic Fatigue
Syndrome: A Critical Review
Adam J. O’Neal and Maureen R. Hanson*
Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex,
multi-system disease whose etiological basis has not been established. Enteroviruses
(EVs) as a cause of ME/CFS have sometimes been proposed, as they are known agents
of acute respiratory and gastrointestinal infections that may persist in secondary infection
sites, including the central nervous system, muscle, and heart. To date, the body of
research that has investigated enterovirus infections in relation to ME/CFS supports an
increased prevalence of chronic or persistent enteroviral infections in ME/CFS patient
cohorts than in healthy individuals. Nevertheless, inconsistent results have fueled a
decline in related studies over the past two decades. This review covers the aspects
Edited by: of ME/CFS pathophysiology that are consistent with a chronic enterovirus infection
Eliana Mattos Lacerda,
University of London, United Kingdom
and critically reviews methodologies and approaches used in past EV-related ME/CFS
Reviewed by:
studies. We describe the prior sample types that were interrogated, the methods used
Antonio Gennaro Nicotera, and the limitations to the approaches that were chosen. We conclude that there is
University of Messina, Italy considerable evidence that prior outbreaks of ME/CFS were caused by one or more
Brett Lidbury,
Australian National University, Australia enterovirus groups. Furthermore, we find that the methods used in prior studies were
*Correspondence: inadequate to rule out the presence of chronic enteroviral infections in individuals with
Maureen R. Hanson ME/CFS. Given the possibility that such infections could be contributing to morbidity
mrh5@cornell.edu
and preventing recovery, further studies of appropriate biological samples with the latest
Specialty section:
molecular methods are urgently needed.
This article was submitted to
Keywords: myalgic encephalomyelitis, chronic fatigue syndrome, enterovirus, chronic infection, RT-PCR, serology,
Infectious Diseases - Surveillance,
immunohistochemistry, cell culture
Prevention and Treatment,
a section of the journal
Frontiers in Medicine
INTRODUCTION
Received: 30 March 2021
Accepted: 26 May 2021
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex multi-system disease
Published: 18 June 2021
of unknown cause for which there is little insight into the molecular basis of disease progression,
Citation: persistence and in rare cases - remission. The ME/CFS literature includes findings of patient
O’Neal AJ and Hanson MR (2021) The
immune system irregularities, abnormal cellular energy metabolism, and various altered autonomic
Enterovirus Theory of Disease Etiology
in Myalgic Encephalomyelitis/Chronic
nervous system manifestations including post-orthostatic tachycardia syndrome, orthostatic
Fatigue Syndrome: A Critical Review. intolerance, and dysregulated hypothalamus pituitary adrenal axis. A hallmark symptom, required
Front. Med. 8:688486. for many case definitions, is exercise intolerance or post-exertional malaise (PEM) (1, 2). The name
doi: 10.3389/fmed.2021.688486 of the illness itself is controversial, with one view holding that Myalgic Encephalomyelitis, a name
Frontiers in Medicine | www.frontiersin.org 1 June 2021 | Volume 8 | Article 688486
O’Neal and Hanson Enteroviral Infection in ME/CFS
dating from a series of early outbreaks of the disease (3), defines tests available at the time could not convincingly implicate a
an illness that is different than Chronic Fatigue Syndrome, a specific culprit.
name created in 1988 through a U.S. government committee (4). Subsequent outbreaks displayed the same basic features of
A discussion of the case definition and nomenclature is outside the 1934 outbreak with some distinct clinical presentations
of the scope of this article, so we will use “ME/CFS” despite depending on the region (3, 11, 13). Overall, most epidemic
of the possibility that the initial CFS case definition results in outbreaks have occurred in mid-spring through early fall
inclusion of individuals who would not have met earlier criteria indicating a virus with seasonal epidemic trends may be involved.
for Myalgic Encephalomyelitis. Seasonality is not rare for viruses; many types, including but
ME/CFS case documentation shows evidence of both not limited to echovirus, coxsackievirus and poliovirus-related
sporadic events involving singular individuals and regional species, are well-known to have strong outbreak seasonality
outbreaks involving significant fractions of affected communities, peaking in the month of August or early fall (10, 14). Outbreaks
especially hospitals, schools, and military bases. Machine learning occurring after 1934 that deserve notable mention based on
estimation of ME/CFS prevalence using large-scale medical similar clinical presentations and links to an enteroviral culprit
claims data gives a frequency of diagnosis in the United States are highlighted below:
that falls somewhere between 1.7 and 3.38 million Americans
• 1949–1953 Adelaide, Australia: Dr. R. A. Pellew conducted
(5) and world-wide, the prevalence may be as high as 65
several animal studies using patient throat washings, feces
million (6). ME/CFS is not a rare disease and therefore
and cerebrospinal fluid collected from the 1949–1953 Adelaide
understanding of disease pathophysiology and discovery of
outbreak as inoculants into rhesus monkeys, rabbits, mice,
standardized biological markers or tests are important to identify
and hen eggs. Investigation into two monkeys repeatedly
appropriate treatments.
inoculated with patient sample revealed minute red spots
The pattern of transmissibility, and acute symptom
along the course of the sciatic nerve, infiltration of
constellation reminiscent of a flu-like illness, led early
lymphocytes and mononuclear cells into nerve roots and
investigators to hypothesize a viral theory of ME/CFS disease
nerve fibers showing patchy damage to the myelin sheath
etiology. Indeed, a number of researchers have interrogated
with axon swelling. Although similar to poliovirus inoculation
a diverse range of microbial pathogens as triggers and/or
outcomes, these monkeys displayed more widespread changes
perpetuators of the ME/CFS disease state. These include but are
in additional areas of the nervous system with no evidence
not limited to Epstein-Barr virus, cytomegalovirus, parvovirus
of damage to nerve cells in the brain and spinal cord.
B19, Brucella, Toxoplasma, Coxiella burnetti, Chlamydia
Additionally, severe myocarditis was found in one of the two
pneumoniae, human herpesviruses, enteroviruses, human T cell
monkeys studied – myocarditis being most commonly caused
leukemia virus II-like virus, spumavirus, hepatitis C virus, and
by enteroviruses (10, 15).
human lentiviruses (7–9).
• 1948 Akureyri, Iceland: Incidence of over 1,000 cases
Between the 1930s and 1960s, a number of globally
during a 3 month period resulted in the naming of
occurring ME/CFS outbreaks, with a spatiotemporal incidence
“Icelandic disease,” which would later evolve to “benign
coinciding with poliovirus epidemics, appeared under the titles
myalgic encephalomyelitis” (16). Those who fell ill with
of “abortive or atypical poliomyelitis” transitioning to “benign
the disease showed classical viral-type illness onset which
myalgic encephalomyelitis” or “epidemic neuromyasthenia” as
later developed into a systemic form of the illness with
physicians sought a term to describe the symptom profile of
symptoms including low fever and significant muscle
affected individuals (3, 10, 11). A ME/CFS outbreak occurred
tenderness/weakness. Due to the occurrence of concurrent
in 1934 California and provides a representative example
local poliomyelitis epidemics, infectious disease testing was
of clinical features experienced by patients during similar
conducted but failed to indicate poliovirus, coxsackievirus or
epidemics of the time. Briefly, the 1934 outbreak occurred
other known encephalitis viruses (10).
among roughly 200 hospital employees, primarily female,
• 1956 Thorshofn/Egilsstadir, Iceland: Differential poliovirus
who fell ill with what acutely appeared to be poliomyelitis.
vaccination responses between children exposed verses
Epidemiological deviations from what is commonly expected
unexposed to the “Icelandic disease” indicated the etiological
in poliomyelitis epidemics included relatively high attack
agent in ME/CFS may be a virus immunologically related
rates, low mortality rates, low paralytic rates and a high
to poliovirus. Children in a northeastern village of Iceland,
incidence in adults as opposed to young children. Symptoms of
Thorshofn, generated a slight rise in antibody production
sufferers included significant diurnal temperature fluctuations,
following vaccine administration whereas children from
localized muscular weakness as well as pain and muscle
tenderness. Patients further exhibited numbness, paresthesia, Egilsstadir, roughly 200 km south, had a much stronger
exercise intolerance, and recurrent systemic and neurological immune response to polio vaccine administration. The
symptoms. Longitudinal tracking of a subset of these patients difference between the two locations was that children from
showed residual muscle alterations, fatigue, and mental changes. Eglisstadir were from an area which recently experienced a
Electromyograms showed generalized, mild, motor neuron myalgic encephalomyelitis outbreak whereas children from
changes and observations indicated that recurrences could occur Thorshofn were not (17). This indirect evidence of unknown
even after many years of relatively normal health (10, 12). The prior immunity was also noted in the aforementioned
totality of these findings indicated an infectious agent although Adelaide outbreak. This was evidenced by a 43% reduction
Frontiers in Medicine | www.frontiersin.org 2 June 2021 | Volume 8 | Article 688486O’Neal and Hanson Enteroviral Infection in ME/CFS
in polio cases in the south of Australia, where Adelaide BACKGROUND REGARDING
is located, compared to regions such as New South ENTEROVIRUSES
Wales and Queensland that reported increased polio cases
(18). Enteroviral cross-immunity is well-documented in the Enterovirus Classification and Basic
enterovirus field and suggests that children in ME/CFS affected Molecular Biology
areas had been exposed to an agent immunologically similar to Although poliovirus is the most well-known enterovirus, it
poliovirus (19). belongs to only one of 15 total enterovirus species including
enterovirus species A-L and rhinovirus species A-C. Of the true
Similar epidemic events of ME/CFS have occurred globally
enteroviruses, species A-D are known to have caused a wide
over time where patients display acute symptoms are similar
spectrum of severe and deadly epidemics in humans (21, 22).
to some poliomyelitis-afflicted patients. The later phases of
The enterovirus genome consists of a single stranded positive
disease progression make evident several differences between
sense RNA molecule roughly 7.5 kb in length (see Figure 1).
ME patients and those with poliomyelitis. The occurrence of
Upon translation via host cell machinery, one full length
considerable symptom constellation overlaps between ME/CFS,
polypeptide is produced and then proteolytically cleaved into the
poliomyelitis and other non-polio enterovirus-related clinical
polyprotein products PI, P2, and P3. P1 encodes four structural
outcomes as well as similarity in epidemic seasonality is further
proteins, VP1-VP4, forming the non-enveloped virion capsid. P2
circumstantial evidence for a relationship between ME/CFS and
and P3 are proteolytically cleaved into 10 non-structural proteins
enteroviruses. One possibility for the co-occurrence of polio
including 2A to 2C, 3A to 3D as well as precursors 2BC, 3AB, and
and non-polio enteroviral outbreaks may be the environmental
3CD. Viral genomic RNA is capped on its 5′ end with the viral-
source of enteroviruses, which often are contaminated bodies
encoded protein VPg (3B) instead of a methylated nucleotide
of water. If sewage is contaminating water, consumers may be
cap structure.
exposed to multiple types of enteroviruses.
Enteroviruses gain cellular entry through binding to
To date, the body of research investigating enterovirus
host cell receptors and undergoing receptor-mediated
infections in relation to ME/CFS supports an increased
endocytosis. Cellular receptors vary between EVs and include
prevalence of chronic or persistent infections in several ME/CFS
CD155/poliovirus receptor, integrins αvβ6 and αvβ3, ICAM-
patient cohorts. The majority of early EV-related investigations
1, ICAM-5, CD55/decay accelerating factor, KREMEN1,
occurred within the UK from the 1970s to early 2000s, starting
coxsackievirus and adenovirus receptor (CAR), scavenger
with serological tests but advancing to molecular methods
receptor B2, P-selectin glycoprotein ligand-1, sialylated glycan,
including immunohistochemical detection of enterovirus viral
heparan sulfate, neonatal Fc receptor, and annexin II (24–28).
capsid protein (VP1) and viral genome detection using RT-PCR
Upon cellular entry, translation occurs following ribosome
(3, 13). Although a significant number of early papers provided
binding onto a type I internal ribosome entry site (IRES) located
evidence for an association of chronic enteroviral infections with
within the 5′ UTR of the viral genome. Replication occurs via
ME/CFS, research into the enteroviral theory of disease etiology
the viral encoded RNA-dependent RNA polymerase (3Dpol)
largely died out in the early 2000s with a few exceptions (7, 20).
which forms the negative sense RNA complement that is used to
One reason that enteroviral research in ME/CFS has languished is
create additional positive sense RNA genomes (29). During active
the difficulty of detecting virus after time has passed following an
infection the ratio of positive to negative strands is roughly 100:1,
acute infection. Furthermore, because enteroviral infections are
whereas chronic infections display a ratio closer to 1:1 (7).
frequent and common, a large fraction of the population will have
5′ and 3′ UTR secondary structures recruit both viral and host
serological evidence of exposures. Another issue is that reports
cell proteins to aid in viral translation and replication (30). The
of association of other pathogens and environmental stresses led
5′ UTR of EVs contains a cloverleaf secondary structure, termed
to the concept that many different types of insults could result in
Domain 1, as well as an internal ribosome entry site (IRES)
ME/CFS. We are offering a critical evaluation of current literature
containing six major stem-loop structures (see Figure 1). The
that may lead to further inquiry into the role of EVs in ME/CFS.
5′ UTR is required for initiating both negative and positive strand
In this review, we will first cover what is known about
RNA synthesis. The 3′ UTR also contains important secondary
enteroviruses in relation to tissue tropism and ability to persist
structures, two predominant hairpin loops, that are the essential
in a chronic infectious state. Emphasis will be put on the aspects
structure of the origin or replication for negative strand synthesis.
of ME/CFS patient pathophysiology that are consistent with an
Proteins bound to the 5′ UTR interact with others bound to
active, chronic enterovirus infection. We will provide a critical
the genome’s polyadenylation sequence at the 3′ end, thereby
review of studies that were attempting to identify chronic EV
promoting viral genome circularization. Circularization allows
infections. The studies will be categorized based on the research
the 3′ UTR secondary structures to act as the initiation site for
methodology employed and special emphasis will be put on
3Dpol binding and at the origin of replication (31).
the sample types used and limitations to the chosen methods.
The viral encoded RNA polymerase is error-prone due to lack
We hope this review may help guide future viral-related studies of a proof-reading mechanism, resulting in high mutation rates
by highlighting the tissue types and approaches most likely to throughout enteroviral evolution. Furthermore, intra- and inter-
provide insight into the hypothesis that enterovirus infections are typic genetic recombination may occur between enteroviruses,
initiating and/or perpetuating the disease state in ME/CFS. leading to increased genotypic plasticity. Enterovirus genomes
Frontiers in Medicine | www.frontiersin.org 3 June 2021 | Volume 8 | Article 688486O’Neal and Hanson Enteroviral Infection in ME/CFS
FIGURE 1 | Representative enterovirus genome structure with emphasis on 5′ UTR Domain I and genome replication. (A) Graphical depiction of EV genome as well as
proteolytic processing to produce all structural and non-structural proteins. Number ranges indicate nucleotide positions for domains 1–7 in the 5′ UTR of CVB4.
(B) 2D illustration of CVB4 Domain I secondary structure. Numbers indicate nucleotide positions. (C) From (23) by license: Creative Commons Attribution 4.0
International. An integrated model for enterovirus replication. Negative-strand synthesis is initiated by circularization of the positive-strand genome via a protein-protein
′ ′
bridge through the interaction of the ternary complex at the 5 -end (3CD and PCBP bound to the cloverleaf structure) and PABP bound to the 3 -poly(A)tail (I. + II.).
CRE-mediated VPg-pUpU acts as primer of the reaction and the polymerase 3D synthesizes the new negative-strand (III.), resulting in a double-stranded intermediate
′
(RF) (IV.). The positive-negative duplex RNA intermediate unwinds, so that the cloverleaf structure at the 5 -end of the positive-strand can form. 3CD and PCBP bind
′
to the cloverleaf to form a ternary complex, which, in turn, will initiate positive-strand synthesis on the 3 -end of the negative-strand (V.). The primer, VPg-pUpU, is
′
recruited and binds to the 3 -terminal AA of the negative strand, and the new positive-strand is synthesized by the polymerase, 3D (VI.).
frequently exhibit mosaic genomic sequences leading to a wide coxsackie adenovirus receptor (CAR) expression that became
variety of genotypic and phenotypic diversity across enterovirus dominant in culture within several passages (43–45). These
serotypes (32, 33). findings together illustrate the host cell’s influence in the co-
evolutionary balance between host and virus as the host attempts
Enterovirus Carrier-State vs. Steady-State to limit viral infection from spreading via reduction in viral
Persistent Infections entry receptor expression (44). CVB1 infection of PANC-1
Persistent enteroviral infections are generally agreed to occur cells also demonstrates that CVB1 drives downregulation of
in two forms, termed carrier-state and steady-state persistence. cellular proteins involved in mitochondrial energy metabolism.
In carrier-state infections, high levels of infectious virus are Mitochondrial dysfunction, oxidative phosphorylation, fatty
produced with infection limited to only a small proportion acid alpha- and beta-oxidation, citric acid cycle and leucine
of cells. Alternatively, steady-state infections show all cells are and valine degradation pathways were significantly enriched
simultaneously infected but viral replication is slowed, leading among downregulated proteins detected by mass spectrometry.
to a non-lytic phenotype with low viral copy numbers per cell. Interestingly, further investigation into the mitochondrial
Both types of persistent viral infections are known to occur across networks of PANC-1 infected cells revealed differential changes
human enteroviral species and have been linked to multiple in mitochondrial network morphology based on the CVB1
clinical conditions (34–42). (ATCC vs. 10796) strain used to generate carrier-state infections.
Research on CVB4 infections of pancreatic ductal-like CVB1 strain 10796 produced fragmented mitochondrial
cells (PANC-1) and murine cardiac myocytes (HL-1) shows networks whereas uninfected cells or those infected with
productive viral replication (106 -108 PFU/ml) is restricted to CVB1 strain ATCC both showed filamentous mitochondrial
a limited subpopulation of cells in culture and are therefore networks. Proteomic analysis further supported these findings
examples of carrier-state infections in vitro. PANC-1 cells by revealing a significant downregulation in mitochondrial
exhibiting resistance to lysis via subsequent CVB4 superinfection proteins involved in fusion processes including mitfusion-1,
were determined to be those PANC-1 cells with downregulated mitofusion-2, and mitochondrial dynamin-like GTPase OPA1
Frontiers in Medicine | www.frontiersin.org 4 June 2021 | Volume 8 | Article 688486O’Neal and Hanson Enteroviral Infection in ME/CFS
in the strain 10796-induced persistent infection model (46). information on mitochondrial enzymology and mitochondrial
In addition to support for carrier-state coxsackievirus-induced membrane potential is absent (59). Although there is a serious
infections in the pancreas and heart, in-vitro infection of human lack of literature pertaining to enterovirus steady-state infections
astrocyte cells also suggests persistent coxsackievirus infection and mitochondrial dysfunction, persistent Echovirus 6 infections
could occur in the central nervous system (CNS) (47). are associated with non-lytic viral RNA and alterations in capsid
Steady-state infections are characterized by all cells in culture protein production including unprocessed capsid polypeptide
having low levels of non-lytic viral replication. Low levels V0 (49). Considering the large number of interactions between
of viral replication lead to decreased viral-induced inhibition enteroviral encoded and host proteins, it is reasonable to
of host cell protein synthesis and thus lead to the non- assume a downregulation and variation in viral encoded protein
lytic phenotype. To date, multiple studies have shown a production during steady-state infections could lead to a
subset of enterovirus serotypes, including coxsackieviruses and mitochondrial dysfunction phenotype different and less extensive
echoviruses, are able to produce low replicative steady-state than seen in enterovirus carrier-state infections, acute infections,
infections without cytopathic effect. This phenomenon may be or cells without infection.
caused by a number of factors including but not limited to
5′ UTR terminal deletions that lead to replication deficiencies or
reduced type I interferon response elicitation, faulty virion capsid
formation due to incomplete capsid polypeptide processing, and Mitochondrial Abnormalities in ME/CFS
alternative EV RNA mutations that lead to abnormalities such Cells
as stable and atypical double-stranded RNA complex formation There is recent literature that describes differences in immune
that inhibits further viral positive strand synthesis (48–51). cell metabolism between ME/CFS patients and controls (60–
In the context of ME/CFS, 5′ UTR terminal deletions and/or 67). The relevance of these reports to possible dysfunction of
atypical dsRNA complex formation are notable, as they have been mitochondria in tissues and organs is unclear. Immune cells
shown to occur in a proportion of ME/CFS patient cohorts in alter their metabolism while responding to signals indicating a
multiple studies (52–54). In a number of cases, chronic diseases threat is present (68, 69). It is not known whether the altered
with some overlap in symptom constellation with ME/CFS mitochondrial metabolism is due to defective signaling or an
show substantial evidence of disease involvement by persistent appropriate immune response that is present in patients rather
infection EV variants. These chronic diseases include idiopathic than healthy individuals, rather than an actual abnormality.
dilated cardiomyopathy (IDCM) (35), chronic inflammatory Early studies on 50 ME/CFS patient muscle biopsies found
myopathy (36), insulin-dependent diabetes mellitus (37–39), mitochondrial abnormalities described as branching and fusion
post-polio syndrome (40, 41), and chronic CNS inflammation of mitochondrial cristae upon ultrastructural examination in
and lesions (42). For example, one study of EV positive-IDCM addition to swelling, vacuolation, myelin figures and secondary
heart tissue detected a positive to negative strand ratio ranging lysosomes indicating mitochondrial degeneration. The authors
from 2 to 20 (35), while another demonstrated EV-negative concluded their work was the first evidence that ME/CFS may be
to positive strand ratios of 1–5 in infected heart tissue (55). due to a mitochondrial disorder caused by a viral infection (70).
Furthermore, the median viral load in heart tissue was assessed A few years later, right quadricep muscle biopsies from
to be 287 EV RNA copies/µg of tissue. Such a low amount nine ME/CFS patients were assayed via electron microscopy,
presents a significant challenge when trying to detect persistent immunochemistry, mtDNA sequencing (as discussed earlier)
enteroviral infections in difficult- to-sample/invasive secondary and enzyme activity assays. The research group found
tissue screening sites. Low levels of viral replication result in EV mitochondrial structure abnormalities, inversion of the
RNA levels so small that they may be past the lower limit of cytochrome oxidase/succinate dehydrogenase ratio and a
detection (51). reduction in some mitochondrial enzyme activities. The enzyme
In reviewing the outcomes of persistent in-vitro EV infections, activity assay results indicate a reduction of the muscle oxidative
it is clear that EVs with the ability to create carrier-state property evaluated on multiple mitochondrial matrix enzymes
infections are able to produce cellular outcomes that may be including NADHtr, COX and succinate dehydrogenase. A
relevant to ME/CFS pathophysiology in an EV variant-dependent reduction in mitochondrial enzyme activities was supported for
manner. As mentioned above, specific CVB1 variants (CVB1 cytochrome c oxidase and citrate synthetase as well (71).
10796) disturb mitochondrial network morphology and lead Two recent studies found normal mitochondrial oxidative
to a downregulation of proteins relevant to mitochondrial phosphorylation (oxphos) and normal respiratory chain complex
energy metabolism. In regard to EVs that produce steady-state activity compared to healthy controls. However, insight into
infections, Echovirus 6 and Enterovirus 72 (hepatitis A) are mitochondrial oxidative phosphorylation was determined using
both shown to cause persistent steady-state infections in-vitro plasma creatine kinase as a surrogate measure of oxphos in
(48, 56, 57). Echovirus 6 is also shown to cause persistent in- muscle (72, 73).
vivo infections and is associated with neurological disorders Another recent study used extracellular flux analysis in vitro
of encephalitis and meningitis (58). Unfortunately, literature to determine utilization of various substrates by skeletal muscle
surrounding mitochondrial outcomes relating to these two cells from patients vs. controls. This study found that muscle
viruses is bleak at best. Echovirus 6 infection of cultivated cells from ME/CFS patients had reduced oxphos in comparison
monkey kidney cells shows mitochondria retain their shape but to controls when supplied with glucose as a substrate, while
Frontiers in Medicine | www.frontiersin.org 5 June 2021 | Volume 8 | Article 688486O’Neal and Hanson Enteroviral Infection in ME/CFS
no abnormalities were detected when cells were supplied with enteroviruses. For instance, focal enterovirus encephalitis caused
galactose or fatty acids (74). by coxsackievirus A3 is associated with focal hypoperfusion in
Overall, the literature surrounding mitochondrial dysfunction the right frontal lobe that cleared upon patient recovery from the
in ME. CFS patients is suggestive of bioenergetic abnormalities neurotropic enteroviral infection. This example case is largely
that are within the realm of possible cellular outcomes based similar to multiple SPECT studies indicating ME/CFS patients
on the nature of the persistent viral infection. Varied findings have significant hypo-perfusion in regions of the brain consistent
pertaining to mitochondrial function in ME/CFS muscle biopsies with their patient-specific symptoms (87–92).
may be due to sampling bias as latent enteroviral infections There is a diverse spectrum of tropisms for each enterovirus;
within secondary infection sites may not be uniform and some EVs are neurotropic in nature while others may be
therefore discovery of a cellular pathophysiology would only be myotropic. Among human enterovirus families A-D, there
found if the correct tissue location were interrogated. exists a subset of EVs that are known to be neurotropic;
these include EV71, multiple coxsackievirus group A members,
Enterovirus Cell and Tissue Tropism all coxsackievirus group B members, poliovirus and EVD68,
Each enterovirus has a distinct cell and tissue level tropism that among many others. Not surprisingly, different neurotropic
is governed by both host and viral factors, including cellular enteroviruses gain CNS access via alternative strategies and thus
virus receptor availability, tissue-specific activity of IRES on display distinct CNS tissue tropism. For example, poliovirus
viral RNAs, and innate immune antiviral activities such as mainly infects and replicates in motor neurons in the anterior
interferon (IFN) response. Given these conditions, EVs as a horn of the spinal cord, while EV71 primarily targets neuronal
whole display a wide spectrum of cell and tissue tropism leading progenitor cells (NPSCs) and astrocytes (75). NPSC infection is
to a wide array of disease outcomes. The diseases may appear as particularly advantageous for viral dissemination, transmission,
short-duration sicknesses such as the common cold and acute replication, and persistence. For instance, NPSC infection may
hemorrhagic conjunctivitis or may cause more serious diseases expand CNS presence as the infected NPSCs differentiate into
through infiltration into secondary infection sites such as organs, neuronal, astrocyte and oligodendrocyte lineages. Furthermore,
muscle or central nervous system (CNS), causing myocarditis, NPSC migration following differentiation allows access into new
pericarditis, encephalitis, meningitis, pancreatitis, paralysis, and CNS locations, and lastly, EV infection of NPSCs may trigger EV-
death (75). specific genomic changes that allow the virus to persist in a latent
CNS regulation of autonomic nervous system output state due to the quiescent cellular environment of non-activated
occurs through multi-synaptic connections descending from NPSCs or NPSCs that have moved to a neuronal cell fate (93).
the hypothalamus and midbrain to preganglionic neurons in EVs gain access to the CNS through a diverse set of entry
the brainstem and spinal cord. The central autonomic system mechanisms including direct infection of brain microvascular
is further comprised of connections between a multitude of endothelial cells, retrograde axonal transport following muscle
limbic system structures, such as the amygdala and hippocampus, infection, exosomal transport across the blood-brain barrier
to collectively regulate autonomic nervous system (ANS) (BBB), and hitchhiking inside of migratory infected immune cells
outflow (76). The ANS is subdivided into the sympathetic, with BBB privilege (75). Infection outcomes can follow expected
parasympathetic and enteric nervous systems, which act to changes such as halting of host cell cap-dependent translational
control internal body processes such as blood pressure, heart and events and production of cytopathic effects causing tissue lesions.
breathing rates, body temperature, digestion, metabolism, fluid However, EVs may also establish a persistent/chronic infection
retention, production of bodily fluids, urination, defecation, and producing atypical clinical outcomes, as may be the case in
sexual response (77). ME/CFS (75).
ME/CFS patients have a number of pathophysiological traits Several known EV-CNS infections display autonomic
that point to abnormalities in the ANS, including impaired blood dysfunction symptoms reminiscent of those described in
pressure variability, orthostatic intolerance, high prevalence ME/CFS patients. Damage to the ANS is well-documented
and severity of posturalorthostatic tachycardia syndrome following poliovirus infection; postmortem histopathology
(POTS), delayed gastric emptying, impaired thermoregulation routinely demonstrates damage to the reticular formation
in adolescent patients, loss of capacity to recover from acidosis region of the brainstem whether or not the patient displayed
on repeat exercise, abnormal cardiac output and altered brain spinal cord damage or paralysis (94). The reticular formation,
characteristics in a wide variety of brain regions including the a network of neurons located in the brainstem that project
limbic system structures that govern the ANS (1, 78–81). These into the hypothalamus, thalamus, and cortex, plays a role as
altered brain characteristics include reduced cerebral, brainstem, a cardiodepressor that lowers cardiovascular output. Post-
and cerebral cortex blood flow; impaired reciprocal connectivity polio syndrome (PPS) patients exhibit a high prevalence of
between the vasomotor center, midbrain, and hypothalamus hypertension and tachycardia while ME/CFS patients display
regions; increased neuroinflammation across widely distributed high rates of POTS, which is accompanied by drop in blood
brain areas including but not limited to the hippocampus, pressure. The difference in autonomic dysfunction outcomes
thalamus, midbrain and pons; reduced cerebral glucose between ME/CFS and PPS patients may possibly be due to
metabolism, and lower brain glutathione (1, 82–86). Many of infection with genetically distinct EV serotypes with different
the altered brain characteristics seen in ME/CFS patients are neurotropism and thus different clinical manifestations.
similarly reported in clinical cases associated with neurotropic However, white matter brain lesions upon MRI, slowing
Frontiers in Medicine | www.frontiersin.org 6 June 2021 | Volume 8 | Article 688486O’Neal and Hanson Enteroviral Infection in ME/CFS
of electroencephalography outputs, clinical impairment of RMK), rhesus monkey kidney (LLC-MK2), African green
attention, and abnormal hypothalamic pituitary adrenal axis monkey kidney (Vero, BGMK, GMK), Madin Darby canine
function are shared between patients with PPS and those with kidney (MDCK), human diploid cells lines (MRC-5, WI-
ME/CFS (95). Nevertheless, there is a controversy about whether 38, SF), human embryonic kidney (HEK), human embryonic
the reports of excess white matter lesions in ME/CFS patients are fibroblast (HEF), human epithelial carcinoma (HEp-2), and
instead related to age, a misdiagnosis of neurological disorder, human rhabdomyosarcoma (RD) cells (104).
or due to major depression. A quantitative summary of rigorous The guidelines further state the preferred and alternative
data pertaining to white matter lesions in ME/CFS reported no sample types to use in cell culture inoculation depending on the
significant increase in the lesions (96), but studies that use more clinical syndrome noted in patients. Based on the occurrence
advanced neuroimaging methods are needed. of encephalitis and respiratory clinical syndromes in a large
Three ME/CFS post-mortem brain autopsy studies found proportion of ME/CFS cohorts, preferred sample types include
enteroviral genomic RNA and VP1capsid protein in the brain tissue and broncho-alveolar lavage, with alternatively
hypothalamus, brainstem, cerebral cortex, medial temporal lobe, approved sample types, including cerebrospinal fluid (CSF),
lateral frontal cortex, occipital lobe, and cerebellum (97–99). feces, throat swab, oropharyngeal swab, nasopharyngeal swab,
These findings provide additional support that a persistent and rectal swab (104).
EV infection within patient limbic and extra-limbic tissues is
possible and could be driving the ANS dysfunction observed in
ME/CFS patients. Approaches and Limitation of EV Detection
CNS infections by other EVs such as EV71 and the group Strategies Employed in ME/CFS Studies
B coxsackieviruses result in ANS dysfunctions reminiscent Across the enterovirus and virus literature at large, a number
of ME/CFS pathophysiology. EV71 brainstem encephalitis of methodologies are used to detect the presence of enteroviral
occasionally induces symptoms of ANS involvement including infection in patients. In the early years of virus detection,
fluctuating blood pressure, tachycardia or bradycardia, biological approaches such as serological testing and cell culture
hypertension or hypotension and respiratory distress. EV71 methods were employed. Isolation via cell culture requires
CNS-specific clinical manifestations include myoclonic jerk, patient samples to be inoculated into enterovirus-susceptible
polio-like syndrome, lethargy, limb weakness, altered mental cell lines and then examined periodically for the presence of
status, encephalomyelitis, encephalitis, aseptic meningitis, and viral-induced changes such as cytopathic effect (CPE), which is
rhombencephalitis (100, 101). Of these EV71-related ANS/CNS described as cells becoming rounded, refractile and shrinking
clinical manifestations, altered blood pressure regulation, altered before detaching from the cell surface. The identity of the isolated
heart rate regulation, myoclonic jerk, lethargy, limb weakness virus was then confirmed/typed via tests such as neutralization of
and altered mental status are reported in ME/CFS patients, infectivity with serotype-specific antisera or immunochemistry
indicating a large overlap in symptom constellations between using fluorescent antibodies. The main disadvantages to cell
ME/CFS patients and neurotropic EV infections (102, 103). culture are that inoculation depends on quality of the patient
To summarize, some serotypes of EVs exhibit CNS tropism sample and requires variable and sometimes extended time
and have the ability to produce persistent viral infections that periods to allow detection (105, 106). Some enteroviruses,
result in atypical and distinct chronic clinical outcomes. Another especially persistent enterovirus variants, do not produce CPE
complicating factor is the production of EV quasispecies, a in cell culture. Without CPE, screening for viral nucleic acid or
population of EVs with subpopulations that consist of specific protein would be necessary.
genotypic variants, each with genotypically-dependent functional Serological testing is confounded by several factors.
characteristics. The proportion of the different quasispecies in the First, enteroviruses often produce clinical disease before the
overall population dictates infection initiation, progression and appearance of antibodies, making their detection retrospective.
dynamics of clinical presentation (93). Furthermore, enteroviruses and rhinoviruses have extensive
antigenic heterogeneity and lack cross-reacting antigens, so
that many different antigens would be needed to detect anti-EV
DETECTION OF ENTEROVIRUSES antibodies (105–107). Virus antigen detection can be achieved
both by immunohistochemical detection and ELISA. Viral
World Health Organization EV Surveillance antigens such as VP1 exhibit sequence similarity between
Guidelines serotypes, which is an advantage in detection of enteroviruses,
The World Health Organization, in conjunction with the U.S. but also means that serotype identification is not feasible
Centers for Disease Control, have published guidelines for solely from reaction with a VP1 antigen. Commercial labs
enterovirus surveillance that details recommended procedures with serological tests for EVs are far from comprehensive.
for specimen preservation as well as optimal methods for For instance, the Enterovirus IgG/IgA/IgM ELISA kits sold
enterovirus detection and characterization. Although not all via Virotech Diagnostics detects 14 (CVA9, CVA16, CVB2,
human enteroviruses can be propagated in cell culture, the CVB4, CVB5 and Echo 5, 11, 15, 17, 22, 23, 25, 33) of the
guidelines state that multiple attempts should be made across roughly 120 known EV serotypes (108). The Enterovirus
a variety of cell lines including: primary African green, Antibody Panel lab test provided by ARUP Laboratories similarly
cynomolgus or rhesus monkey kidney cells (AGMK, CMK, detects 14 EV serotypes (CVA9, CVB1-6, Echo 6, 7, 9, 11 and
Frontiers in Medicine | www.frontiersin.org 7 June 2021 | Volume 8 | Article 688486O’Neal and Hanson Enteroviral Infection in ME/CFS
30, poliovirus types 1 and 3) although the serotypes differ infections in a cohort of 4 patients, while the 4 fecal studies
slightly (109). Negative detection of EVs via these commercially reported an increased EV infection prevalence in 2 of 4 studies,
available serological tests does not conclusively eliminate the with cohorts ranging from a 22–25% prevalence across patient
possibility of an EV infection. Other companies, such as SERION cohorts (Table 1).
Diagnostics and Immuno-Biological Laboratories also sell Although the prevalence of EV infections in these studies
enterovirus-specific ELISA kits but with the added benefit of was generally shown to be significantly increased compared to
using recombinant antigens. The recombinant antigens are made healthy control cohorts, limitations in patient sample types and
from conserved and subtype specific domains across a subset cell culture models may have led to findings that underrepresent
of human enteroviruses and are therefore likely to demonstrate the prevalence of EV infections in patient cohorts. Of the five
antigens for all known human enteroviruses. These kits have an cell culture studies, one study used only one cell type (115), 3
increased comprehensive nature, but a positive detection cannot studies used two cell types (115–117) and one study used three
reveal exactly which EV serotype is the culprit in question. cell types (118).
A serological method for detection of antibodies to The most comprehensive study, which utilized three cell
enteroviruses that has not yet been employed in ME/CFS culture types, included green monkey kidney cells, RD cells
is the peptide array, which is comprised of tiled peptides and HeLa cells, which together supply a diversity of enterovirus
corresponding to a virus family. Such an array designed to probe receptors including CAR, CD155 and DAF. These cultures
human herpesviruses has been used to compare ME/CFS patients therefore detect a wide diversity of enteroviruses although the
to healthy controls and individuals with other diseases (110). system is still not totally comprehensive. No enterovirus-positive
An enterovirus peptide array was successfully used to detect fecal samples were found within a cohort of 12 ME/CFS patients
antibodies against EV-68 in some samples of cerebrospinal fluid (118) when the triple-cell culture method was used. EVs may
and serum from patients with acute flaccid myelitis (111). be absent in these patients, but lack of detection might also
The most popular detection method for identification be attributed to the presence of an enterovirus that uses an
of enteroviruses is RT-PCR, with amplification directed at alternative receptor as well as the low likelihood of detecting EV
conserved regions of the enterovirus genome, including those infections in the stool samples of chronically ill patients with
encoding the 5′ UTR, 3Dpol and VP1. VP1 is the region of persistent infections in secondary sites such as muscle and brain
choice to conduct enterovirus typing. However, low sequence tissue. Furthermore, the investigators were searching for CPE,
similarity amidst the approximately 120 enterovirus serotypes and EVs present in chronic infections commonly undergo genetic
means that no one primer set is robustly comprehensive so that changes which reduce CPE. An example of the inadequacy of
RT-PCR methods would have a lower chance of identifying novel CPE is a report that inoculated cell cultures were negative for
EV serotypes than unbiased sequencing. RT-PCR experiments CPE production in human fetal lung fibroblast and tertiary
that use primers directed at the 5′ UTR of enteroviruses can be monkey kidney cell cultures but were nevertheless positive upon
problematic if the enterovirus contains mutations within the RT-PCR (119).
primer binding region, as is known to happen during persistent Two studies utilized Hep-2, VERO, and monkey kidney tissue
infection. Traditional RT-PCR approaches have reduced ability cultures for identification of enterovirus from CSF and feces from
to identify novel enteroviruses that could be etiological agents in 4 and 76 patients, respectively. Innes (115) identified enterovirus
new diseases. in 2 of 4 CSF samples and one of 4 feces samples (115). Yousef et
Northern blots using sequences complementary to EV al. (116) found that 17/76 patients tested positive for enterovirus
genomic regions to detect viral RNA in a gel are similarly infection while only 2/30 controls tested positive (116).
confounded by a lack of comprehensiveness, as the probe Studies reporting the absence of enterovirus infections in
sequence might fail to hybridize to EV serotypes that have ME/CFS patient cohorts using tissue culture approaches had
sufficient variation in targeted sequences. For greater sensitivity small sample sizes and incomprehensive cell culture systems.
and breadth, many researchers have instead used an unbiased Small sample sizes along with the fact that EVs harboring
RNAseq approach to detect enterovirus nucleic acids in patient 5′ UTR deletions do not produce CPE means that no definitive
samples. In terms of disadvantages, RNAseq is expensive and conclusion can be made about the absence of EVs from the data
requires significant read depth in sequencing to identify low in these studies. Furthermore, fecal samples usually identify only
copy transcripts among the sea of nucleic acids that are being acute enterovirus infections and not chronic ones that might
sequenced. Capture approaches have been developed to enhance be in secondary infection sites. Nevertheless, some studies that
sensitivity and increase breadth of viral detection (112–114) screened suboptimal sample types with culture methods did find
(Table 1). an increased prevalence of EV infections, which might have been
due to inclusion of patients who were still in the acute phase
of illness.
Critical Review of EV Detection in ME/CFS
by Method Used Serological Testing for EVs
Tissue Culture Reports A wide variety of serological tests for detection of EVs have
To date, ME/CFS studies reporting the use of tissue culture been developed. Studies between the 1970s and late 1990s
for EV detection have used CSF and feces in 1 and 4 studies, that screened for EV infections in ME/CFS patients largely
respectively (115–118). The singular CSF study reported two EV focused on serological testing. The diversity of testing employed
Frontiers in Medicine | www.frontiersin.org 8 June 2021 | Volume 8 | Article 688486O’Neal and Hanson Enteroviral Infection in ME/CFS
TABLE 1 | Compilation of enterovirus-specific ME/CFS studies listed by tissue type and sub-grouped based on EV detection methodology.
Positive studies Total studies Prevalence in positive cohorts
Blood 20 24 8–100%
Serological test 16 20 8–90%
PCR 4 5 18–100%
RNAseq 0 2 N/A
Muscle 8 11 13–53%
PCR 6 9 13–100%
Northern blot 4 4 21–50%
VP1 immunohistochemistry 0 1 N/A
Throat swab 1 1 17%
PCR 1 1 17%
Stomach tissue 2 2 82%
PCR 1 1 37%
VP1 immunohistochemistry 2 2 82%
dsRNA immunohistochemistry 1 1 64%
Heart Tissue 1 1 N/A
PCR 1 1 N/A
Cerebrospinal fluid 1 2 N/A
Tissue culture 1 1 50%
EV IgG ELISA 0 1 N/A
Brain tissue 3 3 N/A
PCR 2 2 N/A
VP1 immunohistochemistry 2 2 N/A
Feces 2 4 22–25%
PCR 0 1 N/A
Tissue culture 2 4 22–25%
Electron microscopy 0 1 N/A
Many studies utilize multiple detection methods resulting in the total number of studies not equaling the number of studies based on each method.
in a total of 20 serological-based ME/CFS studies included Immunohistochemistry to Detect EV Capsid Proteins
neutralization, complement fixation, micro-metabolic inhibition, and dsRNA
ELISA, indirect immunofluorescence, and VP1 antigen detection The enteroviral capsid protein VP1 is commonly used for
tests. In total, 16 of the 20 studies found an increased prevalence identification of enteroviral virions in ME/CFS patient tissues. In
of CVB signals in ME/CFS cohorts with positive findings ranging total, 5 studies have used this technique on a variety of patient
from 8 to 90% compared to the positive findings in healthy sample types, including muscle, gastrointestinal, and brain tissue
control cohorts that ranged from 0 to 65% (Table 1) (115–118, (Table 1, Supplementary Table 1) (20, 54, 98, 99, 137). Of these,
120–135). 4 out of 5 studies identified the presence of VP1 capsid proteins
The vast majority of studies evaluated the presence of in patient tissue. The muscle tissue study did not detect VP1
antibodies directed only against CVB enteroviruses, with a few staining in samples of a cohort of 30 ME/CFS patients, despite
exceptions in which echo30- and echo9-directed IgG antibodies RT-PCR signals that indicated the presence of EV-RNA in 13 of
were screened via ELISA (118). A notable study was performed in the same 30 patients. The authors suggested that the difference
1997, in which neutralization tests for 11 enteroviruses (CVB1-6 in PCR and VPI immunochemistry resulted from persistent but
and echo 6, 7, 9, 11, 30) found that 100 out of 200 tested patients latent enteroviral infection in patient muscle tissues, in which no
had elevated enteroviral titers (136). detectable amount of virion particles were being produced (137).
Although serological testing in ME/CFS cohorts generally The remaining 4 studies showed positive VP1 staining in both
shows an increase in the prevalence of EV antibodies, the gastrointestinal and brain tissues (20, 54, 98, 99). Gastrointestinal
findings often lack clinical specificity as a high prevalence samples exhibited positive staining rate of 82% in two patient
of EV antibodies are found in the general population from cohorts (n = 165, n = 416). Comparative cohorts for these
previous exposure. In a retrospective study, it cannot be known two studies were healthy controls (n = 34) and patients with
whether the enterovirus infection occurred before or after functional dyspepsia (FD) (n = 66), which displayed a positive
ME/CFS disease onset without having paired sera from both VP1 staining rate of 20 and 83%, respectively (20, 99). Both the
time periods. ME/CFS and FD patient cohorts showed dsRNA staining for 64
Frontiers in Medicine | www.frontiersin.org 9 June 2021 | Volume 8 | Article 688486O’Neal and Hanson Enteroviral Infection in ME/CFS
and 63% of patients, respectively (54). Because persistent/chronic studies indicating a lack of EV presence by RT-PCR used primer
EV infections with reduced CPE and viral replication typically sets from methods 1, 7, and 8, which amplify 44, 33, and
have a 1:1 ratio between enteroviral positive and negative RNA 56% of known human enteroviruses, respectively. Therefore,
strands, finding a high rate of dsRNA in patient tissues indicates an EV infection could have been present and simply escaped
the likely presence of persistent enteroviral infections. One detection due to the primer sets employed. One study reported
study found VP1 in fibroblasts of small blood vessels in the 20.8% (n = 48) of the ME/CFS cohort to have detectable EVs
cerebral cortex and in a small fraction of glial cells in brain compared to 0% (n = 29) of controls even though method 5
(98), while another detected VP1 instead in the pontomedullary was used, in which round 2 PCR primers amplify 0% of EVs
junction, medial temporal lobe, lateral frontal cortex, occipital under conservative PCR parameters and 3% of EVs under less
lobe, cerebellum and midbrain (99). conservative parameters. Either that reported primer sequence
does not function as expected in the in-silico PCR or the
Molecular Approaches to Detect EV Infections particular EV that was detected in these patients is one of the
We identified 24 reports of the use of either Northern Blot few able to be observed with method 5 primers. Poor in-silico
(n = 4) (52, 138–140), RT-PCR (n = 18) (20, 53, 97, 99, 117, PCR amplification using method 5 was caused by the primer
118, 132, 134, 137, 141–145) or RNAseq (n = 2) (146, 147) across OL253 (5′ -GATACTYTGAGCNCCCAT-3′ ) used in the second
multiple sample types including blood, feces, muscle, brain, heart, round PCR. First round primers, OL252 and OL68, as well as
gastrointestinal tissue and throat swabs. In a few cases, a single second round primer OL24 had binding rates to the EV serotypes
publication used RT-PCR on multiple sample types; thus, there with only OL253 lacking in-silico hybridization. Overall, RT-PCR
are 20 independent studies amongst the 24 reports. Seventeen experiments with low rates of positive in-silico PCR amplification
of the twenty publications report detection of EVs in patient are strongly correlated with publications indicating insignificant
samples or indicate an increased prevalence of EV infections differences in EV prevalence between controls and patients
compared to control cohorts (Table 1, Supplementary Table 1). (Table 2, Supplementary Tables 2, 3).
The 4 Northern blot studies used muscle tissue biopsies and As mentioned earlier, EVs are known to exhibit mutations in
were all positive for viral RNA, indicating an EV prevalence the 5′ UTR that result in replication deficiencies. Interestingly, all
between 21 and 50% in ME/CFS with control cohorts showing 8 PCR methodologies used primer pairs targeting the 5′ UTR with
a prevalence between 0 and 1% (52, 138–140). The two RNAseq the exception of method 5 whose reverse primers target the VP4
studies were negative for the presence of EV in blood, whether and VP2 genomic regions (see Figure 2). This is an important
or not blood was taken before or after an exercise stress that consideration as patients infected with EV variants exhibiting
exacerbated subject symptoms (146, 147). While RNAseq is a 5′ UTR deletions may not be successfully targeted by the primer
more comprehensive approach to enterovirus detection than sets employed across these PCR methodologies. In conclusion,
Northern blots, these studies cannot be directly compared since PCR studies aimed at identifying EVs in ME/CFS have been
one used muscle tissue and the other assayed blood samples. crippled by the use of incomprehensive primer sets that target
With regard to EV studies that applied RT-PCR methods, potentially deleted portions of the viral genome.
5 of the 17 reports indicated no significant difference in EV
prevalence between ME/CFS and control cohorts. The 5 reports
were performed on peripheral blood leukocytes (132), muscle
tissue (118, 141, 142), and feces (119). A list of all 8 PCR DISCUSSION
approaches/methods, indicating the primer sets employed in
RT-PCR experiments, was first compiled, and then each PCR Multiple aspects of the ME/CFS pathophysiology, especially
set was examined for its effectiveness for detection of all related to autonomic dysfunction, are reminiscent of chronic
117 known EV serotypes (Table 2, Supplementary Tables 2, 3). neurotropic enterovirus-related diseases and clinical outcomes.
In-silico PCR was run with conservative allowances (1 mismatch This fact, in conjunction with the enterovirus-like seasonality of
and no mismatches within 2 base pairs of the 3′ end) as well ME/CFS epidemics, often occurring in spatiotemporal incidence
as less conservative allowances (4 mismatches with mismatches with known poliomyelitis epidemics of the time, gives strong
being allowed on the 3′ end) to give a range of possible justification for the conclusion that enteroviruses have been
experimental results, given that one in-silico PCR experiment etiological agents in ME/CFS outbreaks.
does not likely represent the true in-vitro PCR outcomes. The less Many ME/CFS patients in a variety of studies indicate a
conservative in-silico experiments resulted in predicted binding viral-like illness immediately preceded their ME/CFS symptoms.
to multiple locations, sometimes over 15 locations along an However, surveys also indicate that patients ascribe their onset to
EV genome, and thus were not likely to represent results that a variety of other reasons, including emotional stress, life events,
would be gained from an actual experiment. Examining the recent travel, accidents, toxic substances, or mold (148, 149).
conservative in-silico PCR experiments that used 1 mismatch However, some of these events and exposures could merely
and 0 allowed mismatches within the 3′ end of the primer be coincidental and actually be due to an enteroviral infection
(Supplementary Table 2 indicated methods 1, 3, 5, 7, and 8 are that was unnoticed or very mild, given that many enteroviral
low in their comprehensive nature with 52, 50, 21/0, 39, and infections are asymptomatic (150). The COVID19 pandemic
65 EVs being amplified out of 117, respectively. Interestingly, has made it obvious that persistent symptoms can arise from
four (118, 119, 132, 141) of the five (118, 119, 132, 141, 142) mild or asymptomatic infections (151). Were the existence of
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